The invention relates generally to the field of biological sample preparation and analysis. More particularly, the subject invention relates to a method and apparatus for enhancing the sensitivity of blood cell analysis.
Flow cytometry is a well known technique for qualitatively and quantitatively analyzing a large number of individual cells for a specific cellular marker in a rapid manner. In a typical application, a fluorescent molecular probe that selectively binds to a predetermined cell marker, such as a fluorochrome-conjugated antibody that specifically binds an intracellular or cell surface antigen, is added to a cell sample to be analyzed so that the probe can bind or xe2x80x9cstainxe2x80x9d the cells within the sample that express the predetermined cell marker. The sample is then placed in flow cytometer and illuminated with a light source to enable the fluorescence associated with each cell in the sample to be quantified. The magnitude of fluorescence emitted from a particular cell correlates with the quantity of cell marker on or in that particular cell. By extrapolating this fluorescence data, the relative quantity of specific phenotypic markers expressed by cells in a sample can be rapidly and accurately determined. For an overview of flow cytometric analysis see, xe2x80x9cFlow Cytometry and Sorting,xe2x80x9d Myron R. Melamed, Tore Lindmo, and Mortimer L. Mendelsohn, eds., New York: Wiley-Liss, Inc., (3rd ed., 1995); Shapiro, H. M., xe2x80x9cPractical Flow Cytometry,xe2x80x9d New York: Wiley-Liss, Inc., (2nd ed., 1990).
Sample preparation for flow cytometric analysis is typically performed in a non-automated fashion, wherein a saturating concentration of a cell marker-specific probe is added to a cell sample by manual pipetting, and the mixture is then incubated for a period of time sufficient to allow the probe to bind the cell marker of interest. For analyses where red blood cells might cause interference (e.g., immuno-phenotyping leukocytes), the red blood cells can be removed from the sample using an agent that specifically lyses erythrocytes (for example, a hypotonic solution, ammonium chloride or carboxylic acid). Traditionally, to remove interfering unbound probe from the cell sample prior to flow cytometric analysis, the mixture is washed by adding excess buffer to the mixture, centrifuging the mixture to separate the cells from the buffer, removing the buffer containing the unbound probe, and resuspending the cells in fresh buffer. The washing procedure can be repeated multiple times to further remove any remaining unbound probe. This non-automated technique is advantageous in that it results in a relatively clean sample that contains few interferants (for example, unbound probe or cell debris) which might generate background noise or interference during the flow cytometric analysis. For many applications, however, this non-automated technique is relatively time-consuming, can result in significant cell loss due to one or more wash steps, and exposes the cells to the potentially deleterious effects (for example, activation of enzymatic processes, granule release, cell destruction, high gravity forces produced by centrifugation, etc).
While the foregoing technique is acceptable for infrequent analyses involving a small number of samples, it is less suitable for protocols involving repeated analyses of a large number of samples. A more automated procedure is generally preferred when flow cytometric analysis is employed for clinical diagnostics, high-throughput screening, or the like. For example, in a typical clinical assay where leukocytes are immunophenotyped using flow cytometry, a sample of whole blood is placed into an apparatus that automatically processes the sample prior to analysis. One such apparatus is the COULTER(copyright) TQ-Prep(trademark) Workstation system manufactured by Beckman Coulter, Inc. (Miami, Fla.). After adding a probe to the sample, this apparatus uses computer-controlled devices to automatically add an agent that lyses erythrocytes in the sample and a cell fixing agent (for example, paraformaldehyde). The prepared sample can then be analyzed using a flow cytometer without further processing. This automated technique is advantageous in that samples of whole blood can be prepared for analysis quickly and efficiently.
A drawback of this lysing technique can be encountered in applications requiring a high degree of sensitivity. In such applications, in the absence of a washing step, the automated technique does not remove interferants, such as unbound probe or debris from the lysed erythrocytes from the sample. The high background signal caused by the fluorescence from the unbound probe, non-specific probe binding, and/or autofluorescence from the cells and debris can obscure results generated from the analysis.
Where a fluorescently-labeled antibody is used to analyze a cell sample for a marker present in low quantities, the absence of a washing step can result in high background fluorescence caused by the unbound antibody present in the sample. Thus, if too many unbound fluorescent antibody molecules are present in the sample, the flow cytometer can not distinguish the signal emitted from the antibody-bound cells from the xe2x80x9cnoisexe2x80x9d generated by the unbound antibody. That is, the xe2x80x9cnoisexe2x80x9d in the sample overwhelms the xe2x80x9csignalxe2x80x9d emanating from the cells of interest. To avoid this, the signal to noise ratio in the sample can be improved by removing the interferants by manually washing. An example of manual washing comprises centrifuging the sample to pellet the cells, decanting the interferants contained in the supernatant, and resuspending the cells in fresh buffer. As described above for the non-automated technique, this manual washing is disadvantageous because it is time consuming, causes cell damage, and can result in significant cell loss.
A need therefore exists for an apparatus and method for quickly and efficiently removing interferants from a cell sample prior to analysis. In addition, the apparatus and method should minimize the risk of exposure to infectious blood because of operator handling of the blood cell sample. An apparatus that performs the foregoing method with only negligible cell loss, and does not expose cells to high gravitational forces or cell packing caused by centrifugation would be especially advantageous.
It has been discovered that filters, such as microporous hollow fiber membranes, can be utilized in cell sample preparation devices to quickly and efficiently remove interferants from a cell sample. More specifically, it has been found that the use of a hollow fiber membrane having a plurality of pores with a mean diameter less than the diameter of the cells of interest can be utilized to remove interferants from a cell sample to improve the signal-to-noise ratio in a cellular assay. Application of vacuum to the hollow fiber membrane permits interferants to be removed from a blood cell sample within a lumen of the filter with little or no cell damage. As the cells themselves do not pass through pores of the membrane, compared with conventional continuous filtration devices, clogging of the filter is less frequent, and cells are exposed to less deleterious forces. Filters within the invention can be installed in a cell processing apparatus such that a blood cell sample can be washed and analyzed automatically.
Accordingly the invention features an apparatus for automatically removing interferants from a sample containing cells. The apparatus includes a vacuum source; a filtration device comprising an impermeable housing that forms an extramembrane chamber wherein said chamber contains a filter that selective retains cells of interest while allowing interferants to pass through the filter, and wherein said housing contains at least three port and wherein at least one port is connected by a conduit to the vacuum source; a conduit from one of said ports in said housing which is adapted to aspirate a cell sample from a sample container into the filtration device by said vacuum source; and a conduit from one of said ports in said housing which fluidly connects to a buffer reservoir, which provides a means for buffer to enter into said filtration device to recover the retained cells through one of said ports.
In a preferred embodiment, the apparatus for automatically removing interferants from a sample containing cells includes a sample container holder adapted for holding a sample container containing the sample of cells; a filtration device comprising a filter that selectively retains the cells while allowing the interferants to pass therethrough; at least one conduit fluidly connecting the sample container to the filtration device whereby the sample can move between the sample container and the filtration device; and a means for recovering the cells from the filtration device. The filter of the apparatus preferably includes a microporous hollow fiber membrane having a plurality of pores sized such that cells are prevented from passing therethrough. For example, the pores can have a mean diameter of between about 0.1 and 5.0 microns. In preferred versions of the apparatus, the microporous hollow fiber membrane is fashioned into at least one tube defining a lumen, the tube having a first port providing a first opening in the tube, and a second port providing a second opening in the tube. In this preferred embodiment, the conduit can be fluidly connected to the at least one lumen via the first port such that the cell sample can be moved from the cell sample container through the first port into the at least one lumen. The second port can be fluidly connected to a buffer reservoir containing a buffer and also fluidly connected to a detergent solution reservoir containing a detergent solution. The means for recovering the cells from the filtration device can include a fluid pump that can be in fluid communication with a buffer reservoir suitable for housing a buffer so that the fluid pump can cause the buffer to flow from the buffer reservoir into the filtration device. In variations, the fluid pump can also cause the buffer to flow from the filtration device into the at least one conduit.
In another aspect of the apparatus of the invention, the filtration device can also include an impermeable housing that forms an extramembrane chamber between the impermeable housing and the microporous hollow fiber membrane. A vacuum source can be fluidly connected to the extramembrane chamber such that application of a vacuum from the vacuum source to the extramembrane chamber causes the sample of cells to be aspirated from the cell sample container through the at least one conduit into at least one lumen of the microporous hollow fiber membrane via the first port, and a portion of the sample of cells to flow through the microporous hollow fiber membrane into the extramembrane chamber.
The apparatus can also include one or more pumps and a plurality of valves. The pumps provide a hydraulic force for transporting the buffer from the buffer reservoir into the lumens of the microporous hollow fiber membrane, and the detergent solution from the detergent solution reservoir into the lumens of the microporous hollow fiber membrane; and the plurality of valves being adapted to open and close such that the vacuum from the vacuum source can be applied to the extramembrane chamber such that the buffer, the detergent solution, and portions of the sample of cells within the lumens of the microporous hollow fiber can be controllably aspirated from the lumens to the extramembrane chamber; and the hydraulic force provided from the pumps can be directed to transport the buffer from the buffer reservoir to the lumens, the buffer from the buffer reservoir to the cell sample container, and the detergent solution from the detergent solution reservoir to the lumens. In another aspect, the apparatus of the invention can include a computer controller for controlling the pumps and valves.
The invention also features a cell analyzing apparatus that includes both a cell washer for removing interferants from a sample of cells and a cell analyzer for analyzing the sample of cells. The cell washer is describe above and the cell analyzer can be any cell analyzers. Preferably the cell analyzer measures fluorescence, such as a flow cytometer.
Also within the invention is an automated method for removing interferants from a sample containing cells. This method includes the steps of applying a vacuum force to a blood cell sample in a first sample container to cause the blood cell sample to contact a filter; applying a force to the blood cell sample in contact with the filter, whereby interferants in the blood cell sample pass through the filter while the cells in the blood cell sample do not pass through the filter; and recovering the cells from the filter. In this method, the filter can include a microporous hollow fiber membrane having a plurality of pores sized such that the cells are prevented from passing therethrough.
The invention also features an automated method of analyzing a phenotypic marker on cells within a sample. This method includes the steps of adding at least one reagent that reacts with blood cells to a blood cell sample to form a test sample mixture; automatically removing interferants from the test sample mixture to yield a washed blood cell sample; and analyzing the washed blood cell sample to determine characteristics of the blood cells. The at least one reagent can be an antibody that specifically binds the phenotypic marker and the antibody can include a fluorescent test label. The step of automatically removing interferants from the test sample mixture can remove greater then 50% of the interferants from the test sample mixture.
The method can also further include the step of lysing erythrocytes in the sample of cells to be analyzed, and/or the step of quantifying the amount of probe bound to the cells in the test sample mixture by use of a flow cytometer.